Summary of Work: The aim of these studies is to determine the mechanisms that regulate developmental expression of chauvinist genes and the roles of the proteins they encode in spermatogenic cells. These proteins have sequences that are either unique or homologous but not identical to proteins in other cells. A homologous protein is either encoded by a gene family member expressed only in spermatogenic cells or by a spermatogenic cell-specific alternative transcript. Nearly fifty chauvinist genes have been identified and these studies use gene knock-out approaches to examine the role of some that are believed to be important in male gamete development and function. Fertilin b is a sperm surface glycoprotein and a member of the ADAM (a disintegrin and metalloprotease domain) family. Collaborators (Myles and Primakoff) have suggested that it has a key role in sperm-egg interaction and binds to an a1B6 integrin on the egg surface. Mice heterozygous for a targeted mutation in the fertilin b gene are being mated and homologous males are expected to have reduced fertility. Protamine 1 and 2 are highly basic nuclear proteins that replace the histones and are thought to be essential for DNA compaction in the absence of nucleosomes during sperm development. They have been shown by a collaborator (Hecht) to have developmentally and transcriptionally regulated expression in spermatids. Chimeric mice have been produced that carry a targeted mutation in the protamine 1 gene and are being mated to produce heterozygous offspring. A knock-out construct for protamine 2 has been completed and will be used to target this gene in embyronic stem cells. It is expected that disruption of one or both genes will lead to abnormal nuclear compaction and infertility in homozygous males. Glyceraldehyde 3-phosphate dehydrogenase is a key glycolytic enzyme and a homolog (GAPD-S) synthesized only in spermatids has a key role in regulating the generation of ATP required for fertilization. The enzyme is also the likely target for (S)-3-chlorolactaldehyde, a male reproductive toxicant that is a metabolite of the industrial solvent epichlorohydrin. Studies with transgenic mice determined that sequences required for correct expression of Gapd-s are within 230 bp of the transcription initiation site. A knock-out construct for Gapd-s is being prepared and the predicted phenotype of the knock-out is normal appearing sperm that are unable to activate glycolysis, achieve hyper-activated motility, or fertilize eggs.